Summary of Study ST003962

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002482. The data can be accessed directly via it's Project DOI: 10.21228/M8TC38 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003962
Study TitleMeasuring TCA cycle directional fluxes using 13C5-glutamine
Study SummaryThis study investigates the directionality of TCA cycle flux at Complex II in various cell lines exhibiting OXPHOS dysfunction using targeted GS-MS analysis. Quantification of the ratio of M+4 and M+3 isotopomers of succinate and fumarate can be achieved via GC-MS and an MRM quantification strategy. This allows for the calculation of the net direction of the TCA cycle at Complex II in the cell lines examined. We observed a decrease in succinate oxidation of ~60% in HEK293T cells treated with Antimycin A compared to untreated cells, consistent with a significant reduction in forward SDH activity. Subsequent loss of SDHAF2 (i.e. SDHAF2KO treated with Antimycin A) led to a further decrease in succinate oxidation and therefore strong inhibition of forward SDH activity that is rescued by re-introduction of SDHAF2FLAG. In contrast, in the presence of Antimycin A, fumarate reduction did not change upon SDHAF2 loss, suggesting that fumarate reduction under these conditions is non-enzymatic, consistent with previous literature. In addition, We assessed net TCA directionality in 143B cells, our eHAP and HeLa models, and the adenocarcinoma breast cancer cell line MCF7. All four cell lines exhibited net reductive TCA upon Antimycin A treatment.
Institute
University of Melbourne
Last NameRoopasingam
First NameKugapreethan
Address30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
Emailk.roopasingam@unimelb.edu.au
Phone0434297212
Submit Date2025-05-29
Raw Data AvailableYes
Raw Data File Type(s)gqd
Analysis Type DetailGC-MS
Release Date2025-06-11
Release Version1
Kugapreethan Roopasingam Kugapreethan Roopasingam
https://dx.doi.org/10.21228/M8TC38
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002482
Project DOI:doi: 10.21228/M8TC38
Project Title:Complex II assembly drives metabolic adaptation to OXPHOS dysfunction.
Project Summary:During acute oxidative phosphorylation (OXPHOS) dysfunction, the reverse activity of succinate dehydrogenase (Complex II) maintains the redox state of Coenzyme Q by utilizing either fumarate or oxygen as terminal electron acceptors. The tendency for one over another has been suggested to be tissue-specific, but the underlying mechanism and consequence of this is unknown. Using quantitative proteomics to screen a panel of HEK293T knockout cell lines, we identified an increase in SDHAF2 protein, a Complex II assembly factor that enhances the flavination of catalytic subunit SDHA, as critical for metabolic adaptation during OXPHOS stress in HEK293T cells. Loss of SDHAF2 during Complex III inhibition resulted in a reduction in Complex II F-site derived reactive oxygen species (ROS), a severe growth impairment, and a net reductive TCA cycle driven by an inability of mitochondria to support additional Complex II assembly. This in turn leads to use of fumarate as terminal electron acceptor at the cost of a ROS-mediated switch to glycolysis. Cell lines adapted to glycolysis did not accumulate SDHAF2 upon OXPHOS stress and exhibited a net reductive TCA cycle and mild growth phenotypes with or without SDHAF2 being present. Thus, our study reveals how Complex II assembly controls a balance between protection of the Q-pool and ROS-meditated signaling during oxidative stress in cells reliant on mitochondrial OXPHOS.
Institute:University of Melbourne
Last Name:Roopasingam
First Name:Kugapreethan
Address:30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
Email:k.roopasingam@unimelb.edu.au
Phone:0434297212

Subject:

Subject ID:SU004099
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA45279728_143B_A-A_MRM_034_UNK-0021_9042025_21143B_A_A_001 143B_A_A
SA45279828_143B_A-A_MRM__035_UNK-0022_9042025_22143B_A_A_002 143B_A_A
SA45279928_143B_A-A_MRM__036_UNK-0023_9042025_23143B_A_A_003 143B_A_A
SA45280027_143B_WT_MRM_028_UNK-0018_9042025_18143B_WT_001 143B_WT
SA45280127_143B_WT_MRM_030_UNK-0020_9042025_20143B_WT_002 143B_WT
SA45280227_143B_WT_MRM__029_UNK-0019_9042025_19143B_WT_003 143B_WT
SA45280314_HAP_A-A_MRM_001_UNK-0046_16042025_46HAP_A-A_001 HAP_A-A
SA45280414_HAP_A-A_MRM_002_UNK-0044_16042025_44HAP_A-A_002 HAP_A-A
SA45280514_HAP_A-A_MRM_003_UNK-0045_16042025_45HAP_A-A_003 HAP_A-A
SA45280613_HAP_MRM_001_UNK-0042_16042025_42HAP_WT_001 HAP_WT
SA45280713_HAP_MRM_002_UNK-0040_16042025_40HAP_WT_002 HAP_WT
SA45280813_HAP_MRM_003_UNK-0041_16042025_41HAP_WT_003 HAP_WT
SA45282104_HEK_A-A_MRM_001_UNK-0014_16042025_14HEK_A-A_001 HEK_A-A
SA45282204_HEK_A-A_MRM_002_UNK-0012_16042025_12HEK_A-A_002 HEK_A-A
SA45282304_HEK_A-A_MRM_003_UNK-0013_16042025_13HEK_A-A_003 HEK_A-A
SA45280905_HEKSDHAF2_MRM_001_UNK-0017_16042025_17HEKSDHAF2KO_001 HEKSDHAF2KO
SA45281005_HEKSDHAF2_MRM_002_UNK-0015_16042025_15HEKSDHAF2KO_002 HEKSDHAF2KO
SA45281105_HEKSDHAF2_MRM_003_UNK-0016_16042025_16HEKSDHAF2KO_003 HEKSDHAF2KO
SA45281206_HEKSDHAF2_A-A_MRM_001_SET1_UNK-0020_20042025_6HEKSDHAF2KO_A-A_001 HEKSDHAF2KO_A-A
SA45281306_HEKSDHAF2_A-A_MRM_001_SET2_UNK-0027_20042025_9HEKSDHAF2KO_A-A_002 HEKSDHAF2KO_A-A
SA45281406_HEKSDHAF2_A-A_MRM_003_SET2_UNK-0026_20042025_8HEKSDHAF2KO_A-A_003 HEKSDHAF2KO_A-A
SA45281508_SDHAF2_CIIIKO_A-A_MRM_001_RERUN_SET2_UNK-0030_20042025_15HEKSDHAF2KO_CIIIKO_A-A_001 HEKSDHAF2KO_CIIIKO_A-A
SA45281608_SDHAF2_CIIIKO_A-A_MRM_001_UNK-0027_16042025_27HEKSDHAF2KO_CIIIKO_A-A_002 HEKSDHAF2KO_CIIIKO_A-A
SA45281708_SDHAF2_CIIIKO_A-A_MRM_003_RERUN_SET2_UNK-0029_20042025_14HEKSDHAF2KO_CIIIKO_A-A_003 HEKSDHAF2KO_CIIIKO_A-A
SA45281807_SDHAF2_SDHAF2FLAG_MRM_001_UNK-0023_16042025_23HEKSDHAF2_SDHAF2FLAG_001 HEKSDHAF2_SDHAF2FLAG
SA45281907_SDHAF2_SDHAF2FLAG_MRM_002_UNK-0021_16042025_21HEKSDHAF2_SDHAF2FLAG_002 HEKSDHAF2_SDHAF2FLAG
SA45282007_SDHAF2_SDHAF2FLAG_MRM_003_UNK-0022_16042025_22HEKSDHAF2_SDHAF2FLAG_003 HEKSDHAF2_SDHAF2FLAG
SA45282403_HEKWT_MRM_001_UNK-0011_16042025_11HEK_WT_001 HEK_WT
SA45282503_HEKWT_MRM_002_UNK-0009_16042025_9HEK_WT_002 HEK_WT
SA45282603_HEKWT_MRM_003_UNK-0010_16042025_10HEK_WT_003 HEK_WT
SA45282720_HeLa_A-A_MRM_001_UNK-0061_16042025_61HeLa_A-A_001 HeLa_A-A
SA45282820_HeLa_A-A_MRM_002_UNK-0059_16042025_59HeLa_A-A_002 HeLa_A-A
SA45282920_HeLa_A-A_MRM_003_UNK-0060_16042025_60HeLa_A-A_003 HeLa_A-A
SA45283019_HeLa_MRM_001_UNK-0058_16042025_58HeLa_WT_001 HeLa_WT
SA45283119_HeLa_MRM_002_UNK-0056_16042025_56HeLa_WT_002 HeLa_WT
SA45283219_HeLa_MRM_003_UNK-0057_16042025_57HeLa_WT_003 HeLa_WT
SA45283326_MCF7_A-A_MRM_001_UNK-0077_16042025_77MCF7_A-A_001 MCF7_A-A
SA45283426_MCF7_A-A_MRM_002_UNK-0075_16042025_75MCF7_A-A_002 MCF7_A-A
SA45283526_MCF7_A-A_MRM_003_UNK-0076_16042025_76MCF7_A-A_003 MCF7_A-A
SA45283625_MCF7_MRM_001_UNK-0074_16042025_74MCF7_WT_001 MCF7_WT
SA45283725_MCF7_MRM_002_UNK-0072_16042025_72MCF7_WT_002 MCF7_WT
SA45283825_MCF7_MRM_003_UNK-0073_16042025_73MCF7_WT_003 MCF7_WT
Showing results 1 to 42 of 42

Collection:

Collection ID:CO004092
Collection Summary:HEK293T. HeLa, eHAP, MCF7 and 143B cells were cultured in either Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) or IMDM media, supplemented with 10 % (v/v) fetal bovine serum (FBS, CellSera), 50 µg/mL uridine (Sigma), and a mixture of 100 µg/mL streptomycin and 100 units/mL penicillin (Thermo Scientific). For stable isotopologue tracing of 13C5-glutamine (Merck), media was replaced with glutamine-free DMEM (Thermo fisher Scientific) or IMDM supplemented with 2 mM of 13C5-glutamine, 1 mM pyruvate (Thermo Scientific), 10% FBS, 50 µg/mL uridine, and a mixture of 100 µg/mL streptomycin and 100 units/mL penicillin.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR004108
Treatment Summary:Relevant cell lines were treated with doxycycline and Antimycin A 24 and 8 hours prior to the addition of tracer media and maintained until the completion of the experiment, respectively.

Sample Preparation:

Sampleprep ID:SP004105
Sampleprep Summary:Polar metabolites were extracted from snap-frozen cells using 600 µL of HPLC grade methanol:chloroform mixture (9:1; [v/v]), along with internal standards (1.66μM 13C5,15N-valine, 1.66μM 13C6-sorbitol), as described above. The clarified supernatants and pooled biological quality controls (PBQC’s) were dried using a CentriVap concentrator (Labconco).

Chromatography:

Chromatography ID:CH004945
Instrument Name:Shimadzu GC-2010
Column Name:Agilent DB-5 GC column (30 m x 0.25 mm, 1 µm)
Column Temperature:100℃
Flow Gradient:N/A
Flow Rate:1 mL/min
Solvent A:N/A
Solvent B:N/A
Chromatography Type:GC

Analysis:

Analysis ID:AN006510
Analysis Type:MS
Chromatography ID:CH004945
Num Factors:42
Num Metabolites:10
Units:Relative abundance
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